HEMATOLOGY 2 LABORATORY: BLOOD COLLECTION (TRANS-1)

ARTERIAL PUNCTURE Complications:

 Puncturing the artery to collect
 Arteriospasm
arterial blood
 Involuntary contraction of the
 Not performed by MEDTECHS
artery
 Only performed by respiratory
 Maybe due to stress or anxious
therapist and physicians
before the procedure
 can be obtained either through a
 Hematoma
catheter placed in an artery, or by
 Or excessive bleeding
using a needle and syringe to
 Prevented by inserting the needle
puncture an artery
without puncturing the vessels
 collecting from artery for analysis of
 Prevented by applying pressure
blood gases (oxygen,c02)
 arterial blood placed in pre-
 Nerve damage
heparinized syringes or directly on
 by choosing an appropriate
iced box
sampling site and avoiding
 test immediately to avoid clotting
redirection of the needle
 can be used to test analytes in the
 what makes dangerous for the
body and protein levels
arterial puncture
PUNCTURE SITES:  When you keep searching on the
artery it may hit and damage the
Radial artery Brachial Femoral
nerves
artery artery
 Fainting
Most On basilica Same with
 by ensuring that the patient is
common used vein on brachial which
site antecubital hard to locate supine with feet elevated before
fossa beginning the blood draw
Found on 4 collateral  patient must be on lying position
wrist circulation of  Prevented by ensuring that the
blood block patient is supine
There is a Block by
pressure tissue and
ligaments
Skills and
practice is
essential in
performing
this
Performed by
medtechs if (45 degree or 90 degrees angle)
they are
supervised by
SKIN PUNCTURE
immediate
 Collecting capillary blood
staff
HEMATOLOGY 2 LABORATORY: BLOOD COLLECTION (TRANS-1)

 a mixture of capillary, venous, and

arterial blood with interstitial
(tissue) fluid and intracellular fluid
 For small amount of blood
 Used 3rd or 4th finger of non-
dominant hand
 Must be 2-4mm depth of puncture • Sites to avoid
 More nerve ending as compared to o inflamed and pallor areas
antecubital fossa o Cold and cyanotic areas
o Congested and edematous areas
o Scarred and heavily calloused
 Capillary blood area
 mixture of chaos arterial and bit
tissue juices Indications for skin puncture:
 sometimes called peripheral blood
 Test which requires small amount of
blood
• Blood collection that requires small
 Blood typing
amount of blood:
 POCT/ glucose testing
o Blood type
 Blood smear preparation
o Mucous testing
 For Newborn patients
• Newborn: 350ml of blood because they  Geriatrics patients (too old patient)
have little amount of blood compare
adults
• Too much loss of blood for children that
may cause anemia called iatrogenic
anemia

 Iatrogenic anemia-type of anemia

occurs because of excessive loss of
blood during collection

 Indications for Skin Puncture:

• Sites of puncture
o Finger
o Earlobe
o <1yo: lateral portion of the
plantar surface of the heel/toe
Lateral –side portion for newborn
Note: Don’t hit iliac to avoid puncturing the
bones which is lot painful
 Advantages of Skin Puncture: Less
tedious
 Order of Draw: for skin puncture!
1. HEMATOLOGY (EDTA)
2. Blood bank tubes
3. chemistry tubes (no-anticoagulant)
HEMATOLOGY 2 LABORATORY: BLOOD COLLECTION (TRANS-1)

 Localized or generalized necrosis

 skin breakdown from repeated use of
adhesive strips (micropore and tapes)

VENIPUNCTURE
 Manner of inserting a needle attached
to a syringe to a palpable vein to
collect blood for laboratory testing
 Specimen collected:
 Most widely/commonly used blood
1. Blood culture sample in all laboratory tests
2. Citrate (blue)  Lot test to performed
3. Non-additive (red)
4. Heparin
5. EDTA Things to remember!
6. Black oxalate

SIR BABY: In skin puncture there also order of
draw because finger is puncture it is normal
process and platelets attracted and go to those
sites to form the clot so there could be
AGGREGATED platelet that can interfere your
testing
Tube should be filled with blood starts with:
1. Proper identification of patient
a. You’ll let the patient state their
own name
b. In Coma state patient you’ll ask the
relative of the patient and let them
state the name of the patient
c. If there is no relative check the id
tag or let nurse identify patient for
you

1. Tubes for Hematology (I.e. EDTA): to prevent 2. Tourniquet application

a) Applied for a maximum of 1 min
unwanted aggregation of blood
beyond that may cause hemolysis
2. Additive tube b) Applied 10-15 cm away to the
puncture site
3. Non additive tub
3. Disinfection
Complications:
a) Disinfect using 70% Alcohol in a
 Collapse of veins if the tibial artery is circular motion (inner to outer)
lacerated from puncturing the medial
aspect of the heel 4. Angle of needle insertion
 Osteomyelitis of the heel bone a) In book suggest for about 25-45
angle
(calcaneus)
b) In real life it depends on the veins
 Nerve damage if the fingers of neonates
of the patient.
are punctured c) If veins is very prominent= 20 or 25
 Hematoma and loss of access to the degree
venous branch used d) Fat or Obese patient= increase
 Scarring angle
HEMATOLOGY 2 LABORATORY: BLOOD COLLECTION (TRANS-1)

 TUBES MUST CONTAIN VACUUM

5. Bevel up  Able to transfer into multiple test tube
a) Hit Skin to vein puncture to prevent in a single puncture
hematoma WINGED COLLECTION
b) SKIN TO VEIN INSERTION  Using a butterfly needle attached in the
syringe
6. Needle length : 1-1.5 inches
Bore:
→ gauge 16 blood donation
→ gauge 21 GREEN
→ gauge 22 BLACK
→ gauge 23 BLUE

7. Position of the patient

a) Supine to seating
b) Position may affect some result
such as increasing potassium etc.
c) Not allow patients to open and
close their fist

8. Label
a) Label them accordingly and after
transfer the blood (name, age,
gender, room, initials of
phlebotomist)
b) We’re not allowed to Relabel

9. Disposal
a) Needle should be in puncture
resistant bottle
b) Yellow bag in infectious material

3 IMPORTANT FACTORS FOR SUCCESFUL

VENIPUNCTURE
1. Phlebotomist
2. Quality of patient’s pain
3. Availability of treatment
SITES OF PUNCTURE

Newborns up to 18 months
METHOD OF COLLECTION: • External Jugular Vein
• Temporal vein
SINGLE COLLECTION *Antecubital fossa (SITE OF CHOICE)
 SYRINGE
 Transfers to a single tubes Older children (18months to 3 yo)
MULTIPLE COLLECTION • Femoral vein
 ETS • Long saphenous vein
HEMATOLOGY 2 LABORATORY: BLOOD COLLECTION (TRANS-1)

• Popliteal vein collected must be discarded because it

• Ankle vein still contains IV fluids
*Antecubital fossa
ADVERSE EVENT:

3yo to adult life

• Wrist vein
• Dorsal vein of hand
• Dorsal vein of ankle Hematoma
*Antecubital fossa  Maybe due to improper collection of
blood or prolonged tourniquet
ANTECUBITAL FOSSA application
 Most ideal site for venipuncture  To prevent it you must apply pressure
to the site
Median- most preferred because it is stable  DO NOT ADVICE PATIENT TO BEND
Cephalic and basilica-unstable and movable so THEIR AR
anchor the vein to collect blood Complications:
• Hematoma
TWO PATTERNS OF VEIN: • Pain
 “H pattern” • Syncope and fainting
 Median capital vein • Iatrogenic anemia
 Cephalic • Infections
 Basilica • Edema
• Allergies
 “M” pattern • Petechiae
 Median vein
 Accessory cephalic Microbiology studies: first draw to conduct
 Basilica *Yellow tube contain gel separator treated like
serum tube.
Sites to Avoid:
• Sites with hematoma- There’s non In transferring the collected blood in a tube:
circulation of blood 1. First way is to transfer it by removing
• Occluded veins the cup of the tube and inject the blood
 impaired blood collection in eye level (Prefer
 Repeat puncturing same 2. . Second is transferring it directly,
syringe to the cup of the tube (before
• Edematous area - Sites with burns, this is not acceptable cause they
scar, tattoo thought it may cause hemolysis but
recent studies prove that it doesn’t
• Sites with Fistula (don’t pass)- After of affect at all
surgery or injury Blood Smear Preparation and Examination

- Blood Smear – accurately identifies the

• IV fluid sites- If it is really necessary to
true condition of the blood/ Depict
collect, you must call the attention of
Picture of true condition of blood
the nurse and let them stop the IV
infusion for about 2-5 mins and after
that the first 5ml of blood that is
Uses of Blood Smear Examination:
HEMATOLOGY 2 LABORATORY: BLOOD COLLECTION (TRANS-1)

● WBC differential count  Blood films in RT >5 hrs often have

- Types of WBC can be accurately unacceptable artifacts (variations in
identified cell)
- Relative count only – represents  Most ideal is disodium or tripotassium
percentage of white cells out of 100 EDTA
cells you identified  EDTA preserves the morphology of cells
- In routine WBC diff. count, only 100  Liquid tripotassium EDTA is preferred
cells counted because it can mix easily with blood.
- Prepared examine for differential count Powdered ones can leave granules even
● Platelet Estimation when mixed
- Platelet count differs depending on the
 Advantage of EDTA:
thickness or thinness of the smear
o Multiple slides can be made if
(subjective)
necessary
- Estimation not accurate
o Generally prevents platelets
- Because distribution of cell within blood
from clumping on the glass slide
smear vary from 1 smear to another
- Heparin as anticoagulant will make the
background bluish
 Thick smear- high cells and clumping of
● Platelet satellitosis/satellitism in vitro
cell
phenomenon that happens in patient’s
 Thin smear-decrease count cells
blood when anticoagulated with EDTA
 We can still examine blood smear to
‒ Pseudothrombocytopenia – platelets
quantify platelets but result must be
adhere to the walls of
obtain on automated machine before
neutrophil/associated to antibodies
 Make sure what u were reporting is the
● Spuriously low platelet count and falsely
equivalent estimate of platelet count
increased WBC counts(pseudoleukocytosis)
 Every platelet count there will be
can result from EDTA-induced platelet
equivalent estimate
clumping
 PCQACL-quality assurance standard in
- Aggregated platelets may be falsely
PHIL. Tells for every CBC there should
identified as wbc
be a platelet count
● To correct satellitism: use sodium citrate as
anticoagulant
● Morphologic evaluation of formed
- Reason of satellitism: associated with
elements
platelet-specific auto-antibodies that
- Abnormal morphologic features must
be noted reacts at RT
- For Proper diagnosis of patients - Use citrate for platelet counting
- Can indicate disease like spherocytic,
schistocyte, achantocytes, giant rbc and - EDTA use for anticoagulant for CBC for
so on cell counting

SPECIMEN FOR SMEAR PREPARATION

Specimen for Smear Examination
Capillary blood sample
EDTA BLOOD
● Finger & heel puncture when preparing the
 Ideally prepared within 2-3 hours from
collection to obtain optimal appearance smear bedside. Use 2nd drop of blood as
of cells. sample
HEMATOLOGY 2 LABORATORY: BLOOD COLLECTION (TRANS-1)
● Advantages: can prepared blood smear bed
side
● Limitations:
- Some platelet clumping must be expected
(still acceptable)
- Only a few films can be made directly
from blood from a skin puncture
- Limited amount to collect from this
PERIPHERAL BLOOD SMEAR PREPARATION
Types of blood smear techniques
1. Wedge technique (double slide/spreader
slide/push smear)
2. Coverslip technique (Ehrlich’s method)
3. Coverslip and slide technique (Beacom’s
method)
4. Automated Slide Making (Spunner’s
method)
Wedge Technique
● The most convenient and commonly used
method for making peripheral blood films
● One slide serves as the film slide, and the
other is the pusher or spreader slide
● Ideal blood Drop: 2-3 mm (not too
thick/thin)
‒ Diff-Safe dispenser – inserted to the
rubber stopper of EDTA tube to help
add drop of blood to the slide
‒ 0.25 inch away from end of the slide
o WBC may accumulate at the
end of the slide
DIFF-SAFE DISPENSER BECAUSE sometimes
amount of blood are too much and too less /
help dispense right amount of blood on blood
smear
- Drop of blood determines the thickness
of blood
- 5. When the slide is held up to the light, the
Four Parameters in making a blood
smear (PASS)
‒ Affects thickness/thinness of
smear
● Pressure
o Low pressure = thick smear
o High pressure = thin smear
● Angle - 30-45°
o Higher = thick smear
o Low = thin smear
● Sample – 2-3 mm blood drop
o Large blood drop = long and thick
smear
o Small blood drop = short and thin smear
● Speed – not too fast and too slow
o Too fast = thicker smear
o Too slow = thin smear
How to properly handle slide:
● Pusher slide must be held securely with
dominant hand in 30-40°. Check for the
consistency of slide. Draw back pusher slide
into the drop of blood
o Do not touch drop of blood if
surface is not smooth
● Let pusher slide touch the blood, wait for
blood to spread out but not reaching the
ends of the slide
● Move the pusher slide forward
Features of a well-made Wedge Peripheral
Blood Film
1. The film is 2/3 to 3/4 the length of the slide.
2. The film is finger-shaped, very slightly
rounded at the feather edge, not bullet-
shaped; this provides the widest area for
examination.
3. The lateral edges of the film are visible.
If blood reach the end of the slides
white cell will accumulate to that
area
4. The film is smooth without irregularities,
holes, or streaks.
thin portion (feather edge) of the film has a
“rainbow” appearance.
6. The whole drop of blood is picked up and
spread. Where cell are equally
distributed
HEMATOLOGY 2 LABORATORY: BLOOD COLLECTION (TRANS-1)
● Well-made peripheral blood Film
- Count on the transition
area of thick and thin;
cells are equally
distributed
● Unacceptable films
- Slide appearance
associated with most
common errors:
To Identify clearly cell
must preserved and not
overlapping or
degenerated
a. Chipped or rough edge on
spreader slide
b. Hesitation in forward motion of spreader
slide
c. Spreader slide pushed too quickly
d. Drop of blood too small
e. Drop of blood not allowed to spread across
the width of the slide
f. Dirt or grease on the slide; may also be due
to elevated lipids in the blood specimen
g. Uneven pressure on the spreader slide
h. Time delay; drop of blood began to dry
AUTOMATED SLIDE MAKING AND STAINING
● Sysmex SP-10 will only conduct
prepare and stain a blood smear
after CBC
● SYSMEX-leading company for
hematologic analyzer
performing CBC
● The system adjusts the size of the drop of
blood used and the angle and speed of the
spreader slide in making a wedge
preparation
● The thickness and thinness of smear
depending on HEMATOCRIT
o If example the Hct is high, smears
are usually thicker and machine will
adjust the smear to make it thinner
● The spreader slide is automatically cleaned
and is ready for the next blood film to
be made
● Films are produced approximately every
30 seconds
● BARCODE:Patient identification
information, such as name, number,
and date for the specimen, is printed on
the slide
Staining of Smears
- Done to easily identify and differentiate
the type of formed elements present in
your blood.
- Helps evaluate morphologic features of
these formed elements
● Purpose: make the cells more visible and to
allow their morphology to be evaluated
● Commonly used stains:
o Wright-Giemsa stain – especially for
malarial cases
o Pure Wright stain
o BOTH polychrome stain that
contain eosin and methylene blue
● Fixative used: Methanol
o Done before staining the slide
o Ensures that the cells are attached
to the slides
o Allow first to dry before staining
● pH of stains: 6.4 – 6.8
o Staining reaction is always pH
dependent
● Buffers used:
o 0.05M sodium phosphate buffer =
6.4 pH
o Aged distilled water = 6.4-6.8 pH
(commonly used)
o To make sure cells to take up stain
and enhance morphologic structure
● Examples:
o Free methylene blue is basic and
stains acidic (and basophilic)
cellular components, such as
ribonucleic acid (RNA)
HEMATOLOGY 2 LABORATORY: BLOOD COLLECTION (TRANS-1)

o Free eosin is acidic and stains basic

(and eosinophilic) components,
such as hemoglobin and
eosinophilic granules Well-Stained Blood Smear
After preparation of smear: (STAIN SMEAR) ● Macroscopically:
‒ Reddish brown : Wright’s stain
1. Fix/immerse slide with
(pink)
methanol for 2-3 mins.
● Microscopically: minimum precipitate with
2. Allow to dry
no artifacts
3. Proceed with staining:
‒ RBC : pink
a. blood smear with Wright or Wright-
‒ Nuclei of leukocytes :purple
Giemsa stain for at least 3 minutes
‒ Eosinophilic granules
4. Add buffer and allow to stay for 3
:red orange
minutes; greenish metallic sheen
‒ Basophilic granules :dark
appearance will be observed
purple
5. Rinse the slide with neutral pH water
‒ Platelets :dark
(distilled water)
lilac
‒ Monocytes: gray ground glass
b. Back of slide is wiped to remove any
appearance
unwanted stain residue
c. Dry smear in a vertical position to make STAINING PROBLEMS
sure no unwanted stains are left on the
slides Too Acidic Stain: Too Alkaline Stain:
d. Prevent air-dry because it may destroy
1. Thin blood smear 1. thick blood smear
morphologic features of cell
2. insufficient 2. prolonged staining
Automated Stainers staining time 3. insufficient
3. prolonged washing
buffering or 4. alkaline pH of
washing stain components
4. old stain Correction :
Correction:
1) check pH
1) lengthen staining 2) shorten stain time
Quick Stains time 3) prolong buffering
2) check stain and time
● Convenient and cost-effective for low-end buffer pH
laboratories 3) shorten buffering
● Fast and easy which takes 1 minute or wash time
● Examine the soonest possible time before
the quality of stain changes
● Modified Wright or Wright-Giemsa stain
● Buffer: aged distilled water
● Stained slides are given a final rinse under a
gentle stream of tap water and allowed to
air-dry
HEMATOLOGY 2 LABORATORY: BLOOD COLLECTION (TRANS-1)
Macroscopic Examination:
‒ Done before microscopic examination
to take note of these possible
appearances in the smear:
● Bluer than normal
- Increased blood proteins and possible
rouleaux
- Associated with multiple myeloma (not
confirmatory)
● Grainy
- RBC agglutination
- Associated with cold agglutinin
syndrome
● Holes all over the smear
- increased lipid levels or sample is
lipemic
● Blue specks at the feathery edge
- markedly increased WBC and platelet
counts
- accumulation of WBC’s
Microscopic Examination
MUST Not too bright not to dark!!!
Proper usage of lenses:
● Light from the illuminator should be
properly centered
● Condenser should be almost all the way up
and adjusted correctly for the magnification
used
● Iris diaphragm should be opened to allow a
comfortable amount of light to the eye
● Scanner & LPO = coarse adjustment knobs
● HPO & OIO = fine adjustment knobs
● PARFOCAL FOCUSING-viewing first
specimen under low magnification to high
magnification
Low Power Objective (LPO 10x)
● Not able to count, identify, and
differentiate cells
● Able to check for overall quality of
smear (if too thick or thin)
● Check for the ff:
- Rouleaux formation
- Color
- Distribution of cells
- Possible to check for the presence
of fibrin strands
- RBC distribution
- Large abnormal cells
● Feather edge and lateral edges should
be checked quickly for WBC distribution
o Accumulation of WBC = repeat
smear
High Power Objective (HPO 40x)
● Select the correct area of the film in
which to begin the differential count
Count where cells are equally
distributed
● Evaluate cellular morphology
● WBC estimate (average # of WBC per
HPO x 20000)
Oil Immersion Objective (OIO 100x)
● Provides the highest magnification
● WBC differential count
● RBC, WBC, and platelet morphology
evaluation
● Platelet estimate
● Differentiate segmented neutrophils
from band cell
● RBC and WBC inclusions
● Reactive or abnormal cells enumeration
● n-RBC counting (if present)
● Cedarwood oil is used
o Add oil in between HPO and
OIO
HEMATOLOGY 2 LABORATORY: BLOOD COLLECTION (TRANS-1)

Optimal Assessment Area o Body

o Tail – thinnest area
● Between the thick area and the very thin
feather edge (heel area)
● IDEAL FIELD:
‒ RBCs are uniformly and singly
distributed(not ‘overlapping), with few
● Examine the smear in between the
touching or overlapping, and have their
body and tail (“heel”)
normal biconcave appearance
1. Fix with methanol (2-3 mins), allow to dry
‒ 200 to 250 RBCs per oil immersion field
2. Filter the stain before transferring it in the
that are not clumping, not disintegrated
coupling jar to remove unwanted artifacts
‒ 200 RBC= 20 platelets or 7-21 platelets
3. Stain the smears either by:
● Too thin area: red cells are distorted, flat
a. Leave in coupling jar
and large
b. Leave on staining rack
● Too thick area: distorted RBC (rouleaux);
Flood the smear with the stain
distorted WBC
4. Wash smear with buffer (d.H2O), allow to
Counting Methods dry
5. Dry the back of slide to prevent unwanted
‒ For counting using a microscope impurities
‒ Never go back to the same field
**Don’t use heparin as an anti-coagulant
● Cross-sectional or crenellation – from side because heparin causes discoloration (bluish)
to side on the background of the smear
● Longitudinal – from the tail toward the
SAMPLE UNDER LPO
head of the smear
● Battlement - Only checks overall quality of smear
- Preferred technique - Cells are overlapping, cell count may
- Uses a pattern of be elevated
consecutive fields
beginning near the tail on a
horizontal edge
- Maintain counting in the optimal area

- Still count where cells are equally

distributed

Procedure in smear preparation:

1. Wear proper PPE

2. Obtain blood sample from specimen, add
drop of blood (2-3 mm) SAMPLE UNDER HPO
3. Allow spreader slide to touch the blood and
spread from side to side then spread it out.
4. Allow to dry
5. Label the smear
● 3 portions of smear
o Head – drop of blood is located
HEMATOLOGY 2 LABORATORY: BLOOD COLLECTION (TRANS-1)
SAMPLE UNDER OIO
PLATELETS
 Smallest formed element in the blood
(7fL in size)
 Play significant role in homeostasis
 Formation of clot
 Stop bleeding by forming clot and plug
 Bone marrow synthesize platelets
Functions:
 Maintenance of vascular integrity and
help repair it during injury
 Essential in the formation of clot
EVALUATING PLATELETS:
1) Quantitative (number of platelets) –
manual (direct and indirect) & automation
2) Qualitative (function of platelets) –
aggregation test and bleeding time
 Difficult to count
* Small size
* Adhesiveness
* Aggregation: blood smears should be
done quickly to prevent aggregation
Quantification of platelets
* Blood smears are not accurate in
counting platelets but can be used to
double check if counts given by auto
analyzers are correct
* Indirect method – still use in laboratory
* Direct method
* Automation- count only 2-20fL – to
identify and count a cell, the machine
base it on the volume of cell. Platelets
are 7fL/more efficient and objective
* Manual- subjective
INDIRECT PLATELET COUNT
 Platelet estimate (not accurate/not actual
reflection number of cell) is performed under
the 100x oil immersion objective lens
 A well-made smear is stained with Wright’s
or Wright’s-Giemsa to visualize cellular
elements of blood including the platelet.
o Thick smear= many cells, cell clumping
o Thin smear= decrease cell count
 Using blood smear the distribution of cell is
vary to one another even it is from single
patient. Also blood smear has no mark/ guide to
count cell
HEMATOLOGY 2 LABORATORY: BLOOD COLLECTION (TRANS-1)

 In an area of the film where the RBCs barely
touch, with a few overlapping, the number of
platelets in 10 oil immersion fields is counted
 On automated machine platelets are low
but on well prepared smear there are lot
of platelets because they were attached to
the wall of neutrophils - PLATELET
SATTELITISM
 USE SODIUM CITRATE TO CORRECT
SATELLISTISM
 Cannot report patient platelets count
solely on direct method because it is just
an estimate it will depend on the
distribution of the cell so mas better yung
automated analyzer and confirm by
indirect method
 Every platelet count computed in indirect
method there is equivalent estimate
Performing a platelet estimate:
1. Select an area of the blood film in which most
RBCs are separated from one another with
minimal overlapping of RBCs.
Well stained blood smear =200 rbc =7-20
platelets
 In instances of significant anemia or
erythrocytosis, use the following formula
for the platelet estimate:
 In differential counting we must count
100 white cell
 And that 100 wbc must achieve
regardless on no. of fields you counted
 COUNT CELL WHERE IT IS EQUALLY
DISTRUBUTED
 To know platelet in a smear move fine
adjustment if there is structure or
granules inside it is PLATELET
 Perfect circle
 color disappear or black if fine
adjustment it was not a PLATELET

2. Using the 100x oil immersion objective, count

the number of platelets in 10 OIO consecutive
fields and calculate the average number of
platelets per field
 Move the fine adjustment from time to time,
3. To obtain the platelet estimate per uL of to verify if it is platelet o has granules o
blood, multiply the average number of platelets Refractile
per field by 20,000
 Artifact: purplish and too dark, becomes black
4. Compare the instrument platelet count with or totally disappears when fine adjustment is
the platelet estimate from the blood film moved
1. Example: if an average of 20 platelets were
observed per 10x oil immersion field, the
platelet estimate is 400,000/uL or mm3
(400x109 /L).

Giant platelets-larger than usual size (Bernard

Soulier/myelodysplastic/may-hegglin)
HEMATOLOGY 2 LABORATORY: BLOOD COLLECTION (TRANS-1)

Anisocytosis-variation in size

DIRECT PLATELET COUNT

- The number of platelets in 1 liter or 1

microliter of blood
- Whole blood (EDTA) is diluted with
Rees-Ecker diluting fluid which makes
platelets readily visible under the
bright-field microscope
- Whole blood (EDTA) is diluted 1:100
with 1% ammonium oxalate to lyse the
non-nucleated RBCs
- Platelets are counted in the central
large square of a hemacytometer
(Improved Neubauer Counting
Chamber)
- Exactly same as WBC AND RBC
COUNTING using hemacytometer
- Use PHASE CONTRAST to enhance
cellular structure and light microscope
(PARFOCAL) use if phase contrast is not
Giant platelets in automation-decrease because
available
they will identify as WBC
MATERIALS NEEDED:
Platelet should be 2-20 FL (PLATELETS)
 Hemocytometer
 Thoma pipet
INDIRECT PLATELET COUNT REPORTING  Suction device
 Thick coverslip
 Cell counter
 Diluting fluids-platelet counting-
isotonic-red cell are still intact

Isotonic
 Rees-Ecker (TONKANTIN METHOD)
diluting fluid: citrate formaldehyde with
NORMAL VALUE: brilliant cresyl blue; dilution factor may
 150 – 400 x 109 /L be change depending on amount to be
used
 150 - 400K/UL BRILLIANT CRESYL -WILL TAKE UP BY
PLATELETS SO PLATELETS WILL SHINING
INDIRECT: AVE X 20K BLUE
 1% ammonium oxalate: dilution is
Ex: every 10 OIO field u count 200 cells= 20 X
always 1:100(CONSTANT); used in
20k=400,000 X 0.001=400X10 x 9/L
unopette system
HEMATOLOGY 2 LABORATORY: BLOOD COLLECTION (TRANS-1)
HEMOCYTOMETER: heart of manual cell
counting
 Primary square: 3mm x 3mm = 9mm2 area o
divided into 9 secondary squares
 Secondary square: 1mm x 1mm = 1mm2 area
o WBC squares: divided into 16 tertiary
square; found on corners
o RBC squares: divided into 25 tertiary
square; has 5 regions
Tertiary square:
o WBC square = 0.0625mm2 area
o RBC square = 0.04mm2 area
o Quaternary square: each RBC square
are further subdivided into 16
quaternary square
o Quaternary RBC square = 0.0025mm2
area
o Vertical & Horizontal lines on the side:
serve as guide or marker
TIPS: Counting platelets can be count on
RBC AND WBC but more preferred on WBC
because it has only 4 secondary squared
compared to the 25 secondary squares of
RBC
PERFORM MANUAL CELL COUNTING
DIRECTLY:
1. Collect blood to EDTA
2. Aspirate blood using thoma until 0.5
mark
3. Wipe tip of pipette
4. Aspirate diluting fluid until 101/11
5. Wipe the tip again
6. Mix for 5 mins
7. Discard 3-5 drops that may contain
excess diluting fluid
8. Charge –coverslip on top and charge
both end making sure no excess fluid
9. Incubate
10. Counting platelets under HPO
WITH REES-ECKER FLUID
A. DILUTION OF BLOOD SAMPLE
1. The capillary bore of the RBC pipette is rinsed
with Rees-Ecker diluting fluid. All excess fluid
should be removed from the pipette before
blood is drawn into the pipette. This step will
ensure platelet will not adhere on the wall of
the capillary bore of the pipette.
2. Draw the blood into the pipette up to 0.5
mark. If excess blood was draw, adjust the
blood level to the exact 0.5 mark with
NSSmoistened cotton. Clean the stem of the
pipette before any adjustment will be made.
3. The blood should be diluted immediately
with the Rees-Ecker solution. Make a 1:200
dilution by filling the pipette with the solution
up to 101 mark. Two pipettes should be use
simultaneously and each should be used to
charge one side of the counting chambers
4. After the dilution, the pipettes should be
shaken immediately for atleast 60 seconds. This
step will prevent platelet clumping and will
ensure accurate counting
HEMATOLOGY 2 LABORATORY: BLOOD COLLECTION (TRANS-1)
CHARGING OF SOLUTION ONTO THE COUNTING
CHAMBER
5. After mixing, discard the first 5 drops from
each pipette.
 Note: do not induce or blot the tip of the
pipette by any absorbent material to facilitate
the process. The drop should drop freely by
gravity alone. Each side of the hemocytometer
should be charge with a different pipette.
6. After charging the chambers, the
hemocytometer should be kept in covered
container for about 10 to 15 minutes with wet
gauze or cotton pads beneath the
hemocytometer to prevent evaporation, this
step will allow the platelets to settle aid in
accurate counting later on.
COUNTING OF PLATELETS
7. Carefully place the hemocytometer on the
microscope stage so as not to disturb the
platelets. Count platelets in 5 RBC squares in
the center of the counting chamber. Both sides
of the counting chamber should be counted and
the results averaged. Using the high power
magnification count all the platelets, they
appear as small, roundish, unevenly shaped
structures.
Note:
 If 5 RBC squares are counted, area
should be 0.22mm2
 If 25 RBC squares (central large
square) are counted, area should be
1mm2
WITH 1% AMMONIUM OXALATE
 Dilution is 1:100 (Unopette or Automatic)
1. Make a 1:100 dilution by placing 20uL of well-
mixed blood into 1980uL of 1% ammonium
oxalate in a small test tube.
2. Mix the dilution thoroughly and charge the
chamber. (Note: a special thin, flat-bottomed
counting chamber is used for phase-microscopy
platelet counts)
3. Place the charged hemacytometer in a moist
chamber for 15 minutes to allow to platelets to
settle.
4. Platelets are counted using the 40X objective
lens (400X total magnification). The platelets
have a diameter of 2 to 4 um and appear round
or oval, displaying a light purple sheen when
phase-contrast microscopy is used. The shape
and color help distinguish the platelets from
highly refractile dirt and debris. “Ghost” RBCs
often are seen in the background
5. Count the number of platelets in the 25
small squares in the center square of the grid.
The area of this center square is 1mm2 .
Platelets should be counted on each side of the
hemacytometer and the difference between the
totals should be less than 10%
6. Calculate the platelet count by using one of
the equations given. Using the first equation as
an example, if 200 plantlets were counted in
the entire center square
Move the fine to know if it is a platelet
or not where the blue color cytoplasm it
will not disappear
Precipitate will disappear and become
dark
HEMATOLOGY 2 LABORATORY: BLOOD COLLECTION (TRANS-1)

NORMAL PLATELET COUNT: 200-400X 109

Low platelet: thrombocytopenia

Blood-70%
Spleen-30%

Abnormal excessive platelets-

THROMBOCYTOPENIA (myelofloritative
disorder: PV, essential thrombocytopenia,
surgery (temporary),bleeding,anemia